The purification of biological materials from uncleared, crude materials or mixtures of materials in a simple, cost effective manner is a great challenge to industry. There are many applications for methods and apparatus which perform such separations, both within the natural and the life sciences. Natural science applications which require purified biological materials include, but are not limited to, the removal of toxic compounds from industrial waste streams, the clean-up of industrial and environmental sites, the detection of contaminants in sewage treatment processes and the like. Among the life science processes which require purified materials are the immunological or other biological assays, biochemical or enzymatic reactions, affinity-based protein purification, DNA/RNA isolation in various molecular biology applications, and cell separations related to cell diagnostics and cell therapeutics.
Separation processes are based on the application of an external force on a mixture, wherein the external force reacts with a property of the components in the mixture. Separation methods can be based on component size (membrane filtration, dialysis, and screening), phase affinity (distillation, chromatography, sublimation, and crystallization), mass (centrifugation and spectrometry) or combinations of properties (gradient transport and electro osmosis).
In one prior art separation method, electrophoresis, an apparatus separates components from a mixture via an electric field. Electrophoresis is the transport of electrically responsive particles in an electric field. Separation is based on different rates of migration of the components through a solution under the influences of an electric field. Particles in a solution create their own ionic environment, hence they create their own electrical field. The electric field of the apparatus causes the components to migrate by interacting with the electrical fields created by the components.
In many molecules, in particular proteins and macromolecules, charge originates from the ionization of functional groups in the molecules. These charged molecules tend to adsorb oppositely charged particles near the phase boundary between the molecule and the solution in which it is placed. This collection of charged particles and the ions adsorbed thereon is referred to as the electrical double layer. The electrical double layer is often larger in size and has a higher charge density than the original particles.
The electrical double layer creates an electric field about the particle. The potential at the surface of the electric double layer, ψO, is defined as Q/(∈a), where Q is the resultant charge on the electric double layer, ∈ is the dielectric constant of the solution and a is the radius of the electrical double layer. This potential of this electric field decreases with distance x according toψx=ψOexp(−κX)where ψO is the value of the potential of the electric field at the surface of the electrical double layer and κ is the Debye-Huckel constantκ=(8πe2n0z2/∈kT)1/2.By definition e is the electronic charge, no is the bulk concentration of each ionic species, z is the valence of the symmetrical electrolyte, T is the absolute temperature and k is the Boltzmann constant.
Upon application of an external electric field to the solution, the particles, including the electric double layer, are subjected to four forces: the electrophoretic attraction K1, the Stokes friction k2, the electrophoretic retardation K3, and the relaxation effect K4.    K1=QE (the product of the charge of the particle and the potential gradient);    K2=−fcU (the product of the electrophoretic velocity, U, and the coefficient of friction of the particle −fe, defined as 6πηa, where η is the viscosity of the solvent);    K3=(∈ζa−Q)E (where ζ is the electrokinetic potential, the electric potential ψ at the shear boundary of the particle).
At equilibrium with the electric field, the sum of these four forces is zero, and the resulting electrophoretic velocity U becomesU=(QE+K3+K4)/fc.
By ignoring the relaxation effect, the electrophoretic mobility μ is determined as the ratio of the velocity over field strength.μ=U/E=(δζ)/(6πη).
As a result the ability of particles to be separated by electrophoresis can be determined from knowledge of the intrinsic properties of the solution and the particles in the solution. Different types of particles will travel through the same solution, while being subjected to the same electric field, at different rates. This rate differentials allow for component separation.
The resolution provided by the rate differential is enhanced if an element of discontinuity is introduced into the electric field. For example, the mixture could be subjected to a pH gradient, the sieving effect of high density gels, or the adherence of one of the substances in the mixture to a supporting medium. Other factors include shape of the separation vessel and gravimetric effects introduced by running the separation in a vertical direction.
Magnetophoresis is the transport of magnetically responsive particles in a magnetic field. A solenoid carrying current generates a magnetic field inside the core of the solenoid which is parallel to the axis of the solenoid. A ferromagnetic core in one half side of the solenoid will generate a magnetic field with the same configuration extending the magnetic field of the pole linearly to the end of the solenoid. The force acting on a particle as a result of the magnetic field is given byF=m·B here m is the magnetic pulse strength of the particle, and B is the flux density (strength) of the magnetic field. Separation is based on different rates of migration of the components through a solution under the influence of the magnetic field. The magnetic field generated by the solenoid causes responsive particles in the solution to become induced magnets, thereby creating their own magnetic field. These particles can be considered to be microscopic magnets. The magnetic field of the apparatus causes the components to migrate by interacting with the magnetic fields created by the components. In magnetophoretic separation devices and methods, the separating force (supplied by the action of an external electric field on the electrical fields of the electrical double layer in electrophoretic devices and methods) is replaced by the action of an external magnetic field on particles exhibiting their own magnetic fields. The components desired to be removed via magnetophoretic devices and methods either exhibit magnetic fields of their own accord or by the attachment thereto of a magnetic particle. Such particles may be iron particles which exhibit magnetic fields by magnetic induction. The components to remain in solution either exhibit no magnetic field or exhibit a weaker a magnetic field and decreased transport properties than that of the desired component.
In the case where none of the components in the solution exhibits its own magnetic field, magnetic particles (with an affinity for the component desired to be separated from the solution) are introduced into the solution. The affinity of the magnetic particles is often the product of having substances (which bind to the component desired to be separated from the solution with greater affinity than for any other components in the solution) placed on the surface of the magnetic particles. The magnetic particles then bind to the component desired to be removed. The usefulness of magnetic particles which have a biological affinity for a substance desired to be removed in such purification processes is well known in the art.
After reactions occur between the substance on the magnetic particle surfaces and the desired component, the particles, with the component bound thereto, are magnetically separated from the solution. The separation from the solution occurs by applying a magnetic field to the solution, thereby causing the magnetic particles, with the component bound thereto, to be transported through the solution toward the point of greatest (external) magnetic field strength. The other components of the solution, having no, or a lesser, magnetic susceptibility are not transported through the solution by the magnetic field. After the magnetic particles, with the component bound thereto, are removed from the solution, the particle-bound component can be recovered from the magnetic particles by methods known in the art for cleaving the bond between component and substance.
A problem exists in the art when one desires to separate components which do not exhibit magnetic fields of their own accord that are significantly different, one from the other in terms of strength and intensity, to allow separation of the components based on the reaction of their own inherent magnetic fields to the external magnetic field. This problem is further exacerbated when the components desired to be separated are of a similar nature in terms of the types of particles which bind thereto. In such instances it is difficult, if not impossible, to produce a magnetic particle with a substance on its surface which will bind to one of the components and not the other. Hence, there is no way to have the components exhibit magnetic fields that are sufficiently different to react differently in response to an external magnetic field, thus allowing the external magnetic field to separate the components. Such a situation arises when one attempts to separate fetal red blood cells from maternal whole blood. As understood herein, “fetal red blood cells” means nucleated fetal red blood cells.
It is desired to obtain a purified sample of fetal blood cells during the gestation period of the fetus. Fetal cells are used to obtain a wealth of information about the gestating fetus. Fetal nucleated blood cells can be used as a source of DNA to determine the fetus gender, and to predict the likelihood of the occurrence of such genetic defects as Down's syndrome, P-thalassemia, phenylketonuria, cystic fibrosis, Duchene's muscular dystrophy, sickle cell anemia, and the like. Known methods of obtaining a purified sample of fetal blood cells, such as taking a periumbilical blood sample (PUBS) expose the fetus to an extremely high risk of injury, and could cause abortion of the fetus. Even amniocentesis, which comprises removing amniotic fluid from the amniotic sac, exposes the fetus to some risk of injury.
It is known in the art that fetal red blood cells are present in the maternal whole blood supply as early as fifteen weeks into the gestation period. Therefore, maternal blood could be a source of fetal red blood cells. Drawing blood from the mother to obtain the supply of fetal red blood cells greatly reduces the risks associated with removing amniotic fluid from the placenta. Hence, it is desirable to develop a method and apparatus for purifying the fetal cell sub-population from the maternal blood sample. The desired method should obtain a high degree of purity while being minimally invasive to the mother and fetus. This would allow the performance of the useful tests on the fetal cells without the risks attendant with the removal of amniotic fluid.
Current devices and methods of separating biological materials, especially those that attempt to separate fetal cells from the maternal blood, are known in the art. All known devices and methods suffer from major drawbacks, among which are 1) the extremely low yield of fetal cells recovered for analysis, 2) the large quantity of maternal blood that is taken, and 3) the large amount of maternal cell contamination that is seen in the fetal blood cell sample, even after the final purification step. Moreover, none of the known devices and methods use the, pulsed, variable strength magnetic fields of the apparatus and method of the current invention.
Magnetic Activated Cell Sorting (MACS) binds small iron beads covered with a monoclonal antibody, specific for the component desired to be removed from the solution. The beads are introduced into the solution, where the antibodies react with the component to be removed, binding the beads to the component. The solution is then applied to a magnetized surface. The magnetic surface attracts the magnetic particles, thereby attracting the component bound thereto. The surface is then washed to remove the non-bead-coated cells. However, when separating fetal blood cells from a maternal blood supply, it has proven difficult to effectively attach iron beads to internal cellular markers on the fetal blood cells (such as fetal hemoglobin). As a result, to date, MACS has failed to provide the high level of separation achieved by the current invention. MACS suffers from non-specific adhesion of components to the metal filings and difficulty in removing magnetized components from the metal filings.
Even in instances where MACS has been performed using surface antigenic markers like CD 71, inherent difficulties in the MACS procedure prevent achieving the high yield and purity from minimal samples as achieved by the current invention. MACS requires a large initial sample to attain appreciable yields of the desired cell sub-population removed from the original sample. The MACS apparatus creates areas having insufficient magnetic field strength to remove the desired sub-population from the sample. Moreover, even when the desired sub-population particles are bound to the MACS apparatus, a large number of washing steps are required to remove the bound cells from the magnetic surface. These washing steps not only decrease the yield of the desired sub-population, but decrease the concentration of the desired sub-population present in the washing step effluent.
Fluorescent Activated Cell Sorting (FACS), an enrichment procedure, uses lasers to excite cells labeled with specific monoclonal antibodies as an enrichment means. These labeled cells are then sorted for further analysis. This labeling method allows one skilled in the art to realize the presence of the desired component in solution, but does nothing to separate the desired component from the solution. Either before of after labeling, the desired substance is removed by other means.
FACS is generally preceded by sieving the maternal blood sample to a discontinuous gradient to form a layer rich in nucleated red blood cells (NRBC). This NRBC layer is then further purified by panning to remove CD 45+ cells (seen on almost all lymphocyte lineage cells, but not seen on NRBC). Markers comprising fluorescently labeled monoclonal antibody are then attached to the remaining cells. Currently, the two markers with the best fetal cell specificity have been monoclonal antibody to the CD 71+ surface antigen and monoclonal antibody to the internal gamma chain of fetal hemoglobin. The fluorescent labeling of the NRBC makes them identifiable for separation from the maternal blood supply by micro manipulators. (Bianchi, 1995).
Cheung et. al. (1996) attempt to isolate fetal cells present in the maternal circulation for genetic screening to search for molecular defects. The method of Cheung et al. comprises the steps of density gradient separation of the maternal blood supply, subjecting the fetal cell enriched layer to MACS utilizing a CD71-binding particle, applying the enriched portion from the MACS to a support, staining with anti-bodies specific for fetal or embryonic hemoglobin, and removing stained fetal cells with micro-manipulators.
U.S. Pat. No. 4,241,176 to Avrameas et al. discloses a magnetic gel for use in separating materials. Optionally the gel can contain an antibody specific to the material which is desired to be separated from a solution. The magnetic gel is placed along the inside walls of a column and held in place by a static magnetic field, as opposed to the current invention which uses pulsed, variable strength magnetic fields.
U.S. Pat. No. 4,375,407 to Kronick discloses a high gradient magnetic separation device having a filamentary magnetic material in the interior chamber thereof. This reference discloses coating the filamentary material with a coating of hydrogel polymer. The device disclosed in this reference uses uniform magnetic fields. There is no teaching of the use of the pulsed, variable strength magnetic fields of the current invention.
U.S. Pat. No. 4,594,160 to Heitmann et al. discloses a magnetic separator having a combination of screens and balls placed in the interior chamber thereof to intensify the magnetic field strength within the chamber. The separator uses direct-current to produce a magnetic field of at least 1.5×105H at all times. Therefore, there is no teaching of pulsing the strength of the magnetic field, as utilized in the current invention.
U.S. Pat. No. 4,772,383 to Christensen also discloses a high gradient magnetic separator having permanent magnetic devices generating strong magnetic fields across a separating chamber, as opposed to the pulsed, variable strength magnetic fields utilized by the current invention.
U.S. Pat. No. 5,004,539 to Colwell et al. discloses a magnetic separator having permanent magnetic elements which cause the separation chamber to be subjected to a permanent magnetic field. The magnetic flux return paths are made of ferromagnetic materials (column 3, lines 13-16), which by definition, permanently maintain their magnetic state. As such, the magnetic flux channels of the separator disclosed in this reference are incapable of producing the pulsed, variable strength magnetic fields utilized by the current invention.
U.S. Pat. No. 5,122,269 to De Reuver discloses a magnetic filter wherein the magnetic gradient across the filter chamber is substantially constant. The substantially constant magnetic gradient is required to maintain even filling, and reduced emptying, of the filter. There is no teaching of using a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,186,827 to Liberti et al. discloses a device and method for separating biological materials using magnets to produce magnetic fields about a contact surface. The magnetic flux density quickly reduces in a direction away from the surface, thereby allowing the device to collect the desired biological component in a thin layer on the contact surface, preventing the undesired component from becoming entrapped in the desired material layer. There is no teaching of the benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,200,084 to Liberti et al. discloses a method and apparatus for magnetically separating biological materials comprising a magnetic field producing element surrounding a chamber which contains magnetic field gradient intensifying means in the form of iron mesh or wires. The desired material is collected on the surface of the magnetic field gradient intensifying means. There is no teaching of the benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,236,824 to Fujiwara et al. discloses an apparatus and method for quantitating the amounts of biological materials separated by high gradient magnetic separation. After separation, a light source, preferably a laser light, is radiated upon the separated material and the amount of returned (scattered, reflected) light is measured to determine the quantity of material separated. There is no teaching of the benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,275,933, to Teng et al. discloses material separation via a discontinuous gradient. Test tubes are filled with three HISTOPAQUE solutions of different densities to provide a triple gradient layer in each of the tubes. Whole maternal blood is added to the top of this triple gradient and the tubes are spun to yield layers into which cells of different densities have been partitioned. The top layer is rich in lymphocytes. The second layer contains NRBCs and the third layer is predominantly populated with granulocytes. As noted at column 6, line 66 through column 7, line 5, the fetal cells are spread about over different layers in the test tube after centrifugation, not in a single discreet layer allowing for easy removal and analysis of the fetal cells. Morever, the number of fetal cells yielded in this method is quite low and varies between different pregnant woman (between 1/10,000 to less than 1/1,000,000). Further, there is no teaching of the benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,279,936 to Vorpahl discloses a method of magnetically separating materials in solution. The materials desired to be separated are bound to magnetic carriers. A second solution, of different density than the material-containing solution is contacted with, without mixing, the material-containing solution. The two fluid system is then subjected to static magnetic fields and the material bound to the carrier migrates across the solution interface. There is no teaching of the benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,340,749 to Fujiwara et al. discloses a method for collecting specimens comprising labeling the specimens with magnetic particles and subjecting the labeled particles to a permanent gradient magnetic field. There is no teaching of the benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,437,987 to Teng et al. (Inventor's name misspelled as “Tens” on the cover page of the patent), is a continuation-in-part of the application which matured into U.S. Pat. No. 5,275,933. The non-magnetophoretic method disclosed in this reference uses a “panning” step after the gradient separation process. After gradient separation, the layer which contains the fetal NRBCs is washed and suspended in a physiologic solution. The solution is then applied to a substrate which has bound thereto cold agglutinin (IgM) antibodies with anti “i” specificity. This antibody binds to an epitope on the fetal NRBCs (made from repeating N-acetyl lactosamine units of a given structure), thereby attaching at least a portion of the fetal NRBCs to the substrate. Non-adherent cells are then washed from the substrate. There is no teaching of the benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,466,574 to Liberti et al. is a continuation-in-part of the application which matured into U.S. Pat. No. 5,186,827. This magnetic flux density of the device disclosed in this reference quickly reduces in a direction away from the surface, thereby allowing the device to collect the desired biological component in a thin layer on the contact surface, preventing the undesired component from becoming entrapped in the desired material layer. The preferred magnetic field generating means are permanent magnets the field strength of which is enhanced through the placement of ferromagnetic materials (permanently magnetized, by definition) in the chamber of the device. There is no teaching of the benefits of a pulsed, variable strength magnetic field as utilized in the current invention.
U.S. Pat. No. 5,639,669 to Ledley discloses a device and method for separating maternal blood cells and fetal blood cells from a mixture comprising the two. The device and method disclosed by Ledley uses ultrasonic mixing to enhance the separation. Although Ledley's method applies an electromagnetic field to a treated sample containing maternal and fetal blood cells, Ledley's method and apparatus differ from those of the current invention. The Ledley method and apparatus do not utilize a pulsed, variable strength magnetic field as utilized in the current invention.
Moreover, Ledley's device and method require manipulation of an extensively pre-treated solution containing the maternal and fetal blood cells to achieve appropriate conditions of O2 concentration, pH, Cl− ion concentration, CO2 concentration and temperature of the solution. Once the optimal conditions are finally reached, the solution is subjected to a magnetic field. To prevent conglomeration of the maternal and fetal blood cells, Ledley utilizes ultrasonic vibrations to aid in the separation process.
International patent Publication No. WO 95/14118 to Shih et al. discloses a method for separating biological materials by electrophoresis using a gellable polymeric material. There is no teaching of a the benefits associated with using a pulsed, variable strength magnetic field to separate biological materials, as utilized in the current invention.